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Vatandoost, J., & Azimifar, M. A. (2019). Expression of Recombinant Factor IX Using the Transient Gene Expression Technique. Research in Molecular Medicine (RMM), 6(2). https://doi.org/10.18502/rmm.v6i2.4538

https://doi.org/10.18502/rmm.v6i2.4538

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authors

  • Jafar Vatandoost
    No author data available.
  • Mohammad Amin Azimifar
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Published Date

  • May 27, 2019

Expression of Recombinant Factor IX Using the Transient Gene Expression Technique

Research in Molecular Medicine (RMM) / Research in Molecular Medicine (RMM): Volume 6, Issue No. 2 /

Abstract

Background: Pilot and large-scale production of recombinant proteins require the presence of stable clones, but the process of selecting stable clones is time consuming. Moreover, continuous clone culturing in large-scale production may cause loss of incoming plasmid and recombinant genes. Considering the advancements in Transient Gene Expression (TGE) technology, the large-scale expression of factor IX (FIX) was investigated in HEK cells by the TGE technique.
Materials and Methods: HEK cells were seeded in a cell factory, and then transfected by pcDNA-hFIX plasmid using calcium phosphate co-precipitation method. Stable HEK-hFIX cells were also seeded in a cell factory, separately. After adding vitamin K, recombinant FIX was quantified in conditioned media using an ELISA. Moreover, its functional activity was assayed using an aPTT test.
Results: The results showed that the expression and activity of FIX by TGE technology was, respectively, 1.6 and 1.5 times higher than that obtained through stable HEK-FIX cells. Since calculating the specific activity revealed that for all time periods it is 0.2 mU/ng, so the increase in activity is due to the increase in the amount of FIX.
Conclusions: HEK cells with higher transfectability seemed to be an appropriate alternative for transient expression for large-scale protein production. Furthermore, if rapid expression of recombinant proteins is intended, TGE can replace costly and low-yield methods.

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